RNA methylation is emerging as a regulatory RNA modification that could have important roles in the control and coordination of gene transcription and protein translation. Herein, we describe an in vivo isotope-tracing methodology to demonstrate that the ribonucleoside 5-methylcytidine (m5C) is subject to oxidative processing in mammals, forming 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C). Furthermore, we have identified hm5C in total RNA from all three domains of life and in polyA-enriched RNA fractions from mammalian cells. This suggests m5C oxidation is a conserved process that could have critical regulatory functions inside cells.

Living organisms respond to environmental changes and xenobiotic exposures by regulating gene expression. While heat shock, unfolded protein, and DNA damage stress responses are well-studied at the levels of the transcriptome and proteome, tRNA-mediated mechanisms are only recently emerging as important modulators of cellular stress responses. Regulation of the stress response by tRNA shows a high functional diversity, ranging from the control of tRNA maturation and translation initiation, to translational enhancement through modification-mediated codon-biased translation of mRNAs encoding stress response proteins, and translational repression by stress-induced tRNA fragments. tRNAs need to be heavily modified post-transcriptionally for full activity, and it is becoming increasingly clear that many aspects of tRNA metabolism and function are regulated through the dynamic introduction and removal of modifications. This review will discuss the many ways that nucleoside modifications confer high functional diversity to tRNAs, with a focus on tRNA modification-mediated regulation of the eukaryotic response to environmental stress and toxicant exposures. Additionally, the potential applications of tRNA modification biology in the development of early biomarkers of pathology will be highlighted.

5-Hydroxymethylcytidine (hm5C) was recently identified as a direct metabolite of m5C in RNA. We investigated the stability of hm5C in human cells using bio-isotopologues and LC-MS/HRMS. This has led to the discovery of a second oxidative metabolite of m5C in RNA, namely 2′-O-methyl-5-hydroxymethylcytidine (hm5Cm). Subsequent quantitative analysis of total RNA from higher organisms revealed varying levels and TET-independent formation of this new RNA modification.

SummaryTo quantify dynamic protein synthesis rates, we developed MITNCAT, a method combining multiplexed isobaric mass tagging with pulsed SILAC (pSILAC) and bio-orthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha), thus enabling high temporal resolution across multiple conditions in a single analysis. MITNCAT quantification of protein synthesis rates following induction of the unfolded protein response revealed global down-regulation of protein synthesis, with stronger down-regulation of glycolytic and protein synthesis machinery proteins, but up-regulation of several key chaperones. Waves of temporally distinct protein synthesis were observed in response to epidermal growth factor, with altered synthesis detectable in the first 15 min. Comparison of protein synthesis with mRNA sequencing and ribosome footprinting distinguished protein synthesis driven by increased transcription versus increased translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins.

Cells respond to environmental stress by regulating gene expression at the level of both transcription and translation. The ∼50 modified ribonucleotides of the human epitranscriptome contribute to the latter, with mounting evidence that dynamic regulation of transfer RNA (tRNA) wobble modifications leads to selective translation of stress response proteins from codon-biased genes. Here we show that the response of human hepatocellular carcinoma cells to arsenite exposure is regulated by the availability of queuine, a micronutrient and essential precursor to the wobble modification queuosine (Q) on tRNAs reading GUN codons. Among oxidizing and alkylating agents at equitoxic concentrations, arsenite exposure caused an oxidant-specific increase in Q that correlated with up-regulation of proteins from codon-biased genes involved in energy metabolism. Limiting queuine increased arsenite-induced cell death, altered translation, increased reactive oxygen species levels, and caused mitochondrial dysfunction. In addition to demonstrating an epitranscriptomic facet of arsenite toxicity and response, our results highlight the links between environmental exposures, stress tolerance, RNA modifications, and micronutrients.